All 3D-SIM images of human cells were acquired on the DeltaVision OMX V3 imaging system (GE Healthcare) with a 100× 1.4 NA oil-immersion objective (Olympus, UPlanSApo), solid-state multimode lasers (488, 405, and 561 nm), and electron-multiplying charge-coupled device cameras (Evolve 512×512, Photometrics). Serial z-stack sectioning was done at 125-nm intervals for SIM mode. The microscope was routinely calibrated with 100-nm fluorescent spheres to calculate both the lateral and axial limits of image resolution. SIM image stacks were reconstructed using SoftWoRx 6.1.1 (GE Healthcare) with the following settings: pixel size, 39.5 nm; channel-specific optical transfer functions; Wiener filter constant, 0.0010; discard negative intensities background; drift correction with respect to first angle; and custom K0 guess angles for camera positions. The reconstructed images were further processed for maximum-intensity projections with SoftWoRx 6.1.1. Pixel registration was corrected to be < 1 pixel for all channels using 100-nm Tetraspeck beads. Images in Figures 4C , 4D , 5B , 6B , 6D , 7A and Supplementary information, Figures S3B , S3C , S8A-D , S9B and S9E are 3D-SIM images acquired on the DeltaVision OMX V3 imaging system (GE Healthcare).
Deltavision omx v3
Manufactured by GE Healthcare
The DeltaVision OMX V3 is a high-resolution, 3D imaging system designed for advanced live-cell and fixed-sample imaging. It employs structured illumination microscopy (SIM) to achieve super-resolution imaging with up to 3.5x improved resolution over traditional widefield microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Uplansapo, by Olympus (4 mentions)
Softworx 6, by GE Healthcare (3 mentions)
Softworx, by Cytiva (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Cultureslide, by BD (1 mentions)
Fv1200 confocal, by Olympus (1 mentions)
2c sted 775 quad scan microscope, by Abberior (1 mentions)
Operetta system, by PerkinElmer (1 mentions)
Tcs sp5, by Leica (1 mentions)
8 protocols using deltavision omx v3
1
High-Resolution 3D-SIM Imaging of Cells
2
Structured Illumination Microscopy Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All SIM images were collected using Deltavision OMX V3 (GE Healthcare Life Sciences). For SIM images, an oil-immersion Plan-Apo 1.4 NA objective and two different lasers (488 nm and 560 nm) were used and their alignments were corrected using multicolor beads before imaging. All SIM images collected using OMX were reconstructed in OMX SoftWoRx (Deltavision). Subsequently, all images were processed in ImageJ.
3
3D Superresolution Imaging with SIM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Superresolution imaging with 3D SIM was performed with a DeltaVision OMX v3 (GE Healthcare) equipped with a 100×/1.40 NA PlanApo oil immersion objective (Olympus), Cascade II:512 EMCCD cameras (Photometrics), and 405-, 488-, and 593-nm diode lasers. Samples were mounted with VECTASHIELD Mounting Medium (Vector Laboratories) and illuminated with coherent scrambled laser light directed through a movable optical grating. Image stacks with 15 images per plane (five phases, three angles) and a z-distance of 125 nm were acquired at ∼23°C and subjected to a computational reconstruction (softWoRX; Applied Precision).
4
Immunofluorescence Staining of CRC Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For staining F. nucleatum and ALDH1 in the CRC tissues, anti‐F. nucleatum antibody was prepared as described previously.[35 ] The antibody was validated for specificity by immunofluorescence. CRC specimens were embedded in O.C.T. and cut into 7 µm‐thick sections. The sections were stained with anti‐ALDH1 and anti‐F. nucleatum antibody, followed by goat anti‐rabbit IgG antibody.
For evaluating lipid droplet‐mediated Numb degradation, the cells were plated on a culture slide (BD Biosciences). After being fixed in 4% PFA and permeabilized in 0.5% Triton X‐100/PBS, the cells were blocked in 5% normal goat serum for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The cells were then stained with 488‐labeled secondary antibody (Life Technologies) for 1 h at room temperature. For lipids or lipid droplets staining, the cells were incubated with Nile red (1 µm ) for 30 min at 37 °C. The slides were observed under the DeltaVision OMX V3 imaging system (Cytiva, GE Healthcare) or a Confocal FV1200 (OLYMPUS). The 3D‐SIM images were reconstructed using a maximum intensity projection of three dimensions. Antibody information including source, catalog number, and dilution is shown in Table S2 , Supporting Information.
For evaluating lipid droplet‐mediated Numb degradation, the cells were plated on a culture slide (BD Biosciences). After being fixed in 4% PFA and permeabilized in 0.5% Triton X‐100/PBS, the cells were blocked in 5% normal goat serum for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The cells were then stained with 488‐labeled secondary antibody (Life Technologies) for 1 h at room temperature. For lipids or lipid droplets staining, the cells were incubated with Nile red (1 µ
5
3D Structured Illumination Microscopy of Caenorhabditis elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Young adult animals were mounted to a drop of M9 containing 1 mg/ml levamisole on a 3% agar pad. The confocal images were taken using micro-Manger and processed by ImageJ. 3D structured illumination microscopy (SIM) images of worms were acquired on the DeltaVision OMX V3 imaging system (GE Healthcare) with a 100×/1.40 NA oil objective (UPlanSApo; Olympus), solid-state multimode lasers (488 and 561 nm), and electron-multiplying charge-coupled device cameras (Evolve 256×256; Photometrics). Serial Z-stack sectioning was done at 125-nm intervals for SIM mode. To obtain optimal images, immersion oils with refractive indices of 1.520 were used for worms on glass coverslips. The microscope is routinely calibrated with 100-nm fluorescent spheres to calculate both the lateral and axial limits of image resolution. SIM image stacks were reconstructed using softWoRx 6.1.1 (GE Healthcare) with the following settings: pixel size, 39.5 nm; channel-specific optical transfer functions; Wiener filter constant, 0.0010; discard negative intensities background; drift correction with respect to first angle; and custom K0 guess angles for camera positions. The reconstructed images were further processed for maximum-intensity projections with softWoRx 6.1.1.Pixel registration was corrected to be less than 1 pixel for all channels using 100-nm Tetraspeck beads.
6
3D-SIM Imaging of Caenorhabditis elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For 3D-SIM images, worms were immobilized with 3 mg/ml levamisole in M9 buffer and transferred to a small glass slide or dish, then covered by a 4–5% (w/v) agar pad. A half hour later, images were acquired on the DeltaVision OMX V3 imaging system (GE Healthcare) with a 100X/1.4NA oil objective (Olympus UPlanSApo), solid-state multimode lasers (488, 405, 561 nm) and electron-multiplying CCD (charge-coupled device) camera (Evolve 512 × 512, Photometrics). Serial Z-stack sectioning was done at 125 nm intervals for SIM mode. To obtain optimal images, immersion oils with refractive indices of 1.520 were used for PVD cell body or branch point on glass coverslips. The microscope is routinely calibrated with 100 nm fluorescent spheres to calculate both the lateral and axial limits of image resolution. SIM image stacks were reconstructed using softWoRx 6.1.1 (GE Healthcare) with the following settings: pixel 39.5 nm; channel-specific optical transfer function; Wiener filter constant 0.0010; discard Negative Intensities background; drift correction with respect to first angle; custom K0 guess angles for camera positions. The reconstructed images were further processed for maximum-intensity projections with softWoRx 6.1.1. Pixel registration was corrected to be less than 1 pixel for all channels using 100 nm Tetraspeck beads.
7
Visualizing IniA Localization in M. smegmatis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pMV261 constructs were transformed into wild-type M. smegmatis mc2155 (Supplementary Table 2 ), and transformants were selected using 20 μg mL−1 carbenicillin and 10 μg mL−1 kanamycin as the antibiotics. Luria-Bertani media was used to culture the bacteria at 37 °C to an OD600 of 0.6–0.8, and expression was induced using 0.2% (w/v) acetamide at 16 °C for 24 h. Cells were harvested and resuspended in PBS buffer and washed three times. A 10 μL aliquot of these cells was stained with 20 μg mL−1 FM4–64 (diluted with PBS using 2 mg mL−1 FM4–64 in DMSO) for 1 min to label the bacterial membrane32 . To obtain optimal images, immersion oils with refractive indices of 1.512 were used for bacterial cells on glass coverslips. 3D-SIM images were acquired on the DeltaVision OMX V3 imaging system (GE Healthcare) with a × 100/1.40 NA oil objective (Olympus UPlanSApo), solid-state multimode lasers (488 nm, 561 nm), and electron-multiplying CCD (charge-coupled device) cameras (Evolve 512 × 512, Photometrics). Serial Z-stack sectioning was done at 125 nm intervals for SIM mode. Three-dimensional reconstructions were performed using IMARIS 8 software (Bitplane AG, Switzerland). IniA and cell membrane surfaces were created using the Surfaces tool with automatic settings based on the fluorescent signals from GFP and FM4–64.
8
Multimodal Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy images were acquired using a Spinning Disk microscope (Perkin Elmer Vox1000) equipped with a × 60 NA 1.4 oil immersion lens (CFI Apochromat TIRF), with a pixel size of 120 nm or with a Leica TCS SP5 confocal microscope using a Plan Apo × 63 NA 1.4 oil immersion objective. Cells were recorded as z-stacks with a z-spacing of 0.2 μm.
Super-resolution microscopy images were acquired using a 3D structured illumination microscope (DeltaVision OMX V3, GE Healthcare) and a 2C STED 775 QUAD Scan microscope (Abberior Instruments). 3D-SIM was performed with a × 100 NA 1.4 objective lens with a pixel size of 39 nm and a z-spacing of 125 nm (ref. 18 (link)). STED was performed with a × 100 NA 1.4 Olympus UPlanSApo objective lens with a pixel size of 20 nm and excitation lasers of 488, 594 or 640 nm, and a 775 nm depletion laser.
High-content imaging was performed using the Operetta system (Perkin Elmer). Samples were imaged using a × 20 NA 0.45 air objective with three planes of 1 μm spacing, using the following filters: DAPI: excitation wavelength (ex): 360–400 nm, emission wavelength (em): 420–480 nm; Alexa488: ex: 460–490 nm, em: 500–550 nm; Alexa594: ex: 560–580 nm, em: 590–640 nm.
Super-resolution microscopy images were acquired using a 3D structured illumination microscope (DeltaVision OMX V3, GE Healthcare) and a 2C STED 775 QUAD Scan microscope (Abberior Instruments). 3D-SIM was performed with a × 100 NA 1.4 objective lens with a pixel size of 39 nm and a z-spacing of 125 nm (ref. 18 (link)). STED was performed with a × 100 NA 1.4 Olympus UPlanSApo objective lens with a pixel size of 20 nm and excitation lasers of 488, 594 or 640 nm, and a 775 nm depletion laser.
High-content imaging was performed using the Operetta system (Perkin Elmer). Samples were imaged using a × 20 NA 0.45 air objective with three planes of 1 μm spacing, using the following filters: DAPI: excitation wavelength (ex): 360–400 nm, emission wavelength (em): 420–480 nm; Alexa488: ex: 460–490 nm, em: 500–550 nm; Alexa594: ex: 560–580 nm, em: 590–640 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!